The examination of cellular samples that have been treated to reveal the expression of biomarkers has long been a valuable tool for biological research and clinical studies. A common treatment has involved the use of antibodies or antibody surrogates such as antibody fragments that are specific for the biomarkers, commonly proteins, of interest. It is typical to directly or indirectly label such antibodies or antibody surrogates with a moiety capable, under appropriate conditions, of generating a signal. One approach has been to attach a fluorescent moiety to the antibody and to interrogate the sample for fluorescence. The signal obtained is commonly indicative of not only the presence but also the amount of biomarker present.
The techniques of tissue treatment and examination have been refined so that the level of expression of a given biomarker in a particular cell or even a compartment of the given cell such as the nucleus, cytoplasm or membrane can be quantitatively determined. Typically the boundaries of these compartments or the cell as a whole are located using well-known histological stains. Commonly the treated cellular sample is examined with digital imaging and the level of different signals emanating from different biomarkers can consequently be readily quantitated.
More recently a technique has been developed which allows testing a given cellular sample for the expression of numerous biomarkers. Generally this technique involves staining the sample with a fluorophore labeled probe to generate signal for one or more probe bound biomarkers, chemically bleaching these signals and re-staining the specimen to generate signals for some further biomarkers. The chemical bleaching step is convenient because there are only a limited number of signals that can be readily differentiated from each other so only a limited number of biomarkers can be examined in a particular step. But with bleaching, the sample may be re-probed and re-evaluated for multiple steps. This cycling method may be used on formalin fixed paraffin embedded tissue (FFPE) samples and cells. Digital images of the specimen are collected after each staining step. The successive images of such a specimen can conveniently be kept in registry using morphological features such as DAPI stained cell nuclei, the signal of which is not modified by the chemical bleaching method.
Another approach has been to examine cells obtained from a frozen tissue specimen by staining them iteratively and bleaching the labels from the previous staining step before applying the next set of stains. The strength of the fluorescent signal associated with each biomarker evaluated is then extracted from the appropriate image.
There have been efforts to utilize this data to identify patterns of biomarker expression. One approach has been to look for such patterns in an entire sample and to binarize the fluorophore signals using a threshold values and generate various expression profiles that are then overlaid on an image of the tissue of interest. However, the data values resulting from the multiplexing and image analysis of cells represents vast biological complexity that may be difficult to analyze. Thus a need exists for a process to enhance the analysis and visualization of the biological data.